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The objective of this study was to investigate the interaction of the nitrogen (N) cycle with methane production in the Florida Everglades, a large freshwater wetland. This study provides an initial analysis of the distribution and expression of N-cycling genes in Water Conservation Area 2A (WCA-2A), a section of the marsh that underwent phosphorus (P) loading for many years due to runoff from upstream agricultural activities. The elevated P resulted in increased primary productivity and an N limitation in P-enriched areas. Results from quantitative real-time PCR (qPCR) analyses indicated that the N cycle in WCA-2A was dominated bynifH andnirK /S , with an increasing trend in copy numbers in P-impacted sites. ManynifH sequences (6 to 44% of the total) andnifH transcript sequences (2 to 49%) clustered with the methanogenicEuryarchaeota , in stark contrast to the proportion of core gene sequences representingArchaea (≤0.27% of SSU rRNA genes) for the WCA-2A microbiota. Notably, archaealnifH gene transcripts were detected at all sites and comprised a significant proportion of totalnifH transcripts obtained from the unimpacted site, indicating that methanogens are actively fixing N2 . Laboratory incubations with soils taken from WCA-2A producednifH transcripts with the production of methane from H2 plus CO2 and acetate as electron donors and carbon sources. Methanogenic N2 fixation is likely to be an important, although largely unrecognized, route through which fixed nitrogen enters the anoxic soils of the Everglades and may have significant relevance regarding methane production in wetlands.IMPORTANCE Wetlands are the most important natural sources of the greenhouse gas methane, and much of that methane emanates from (sub)tropical peatlands. Primary productivity in these peatlands is frequently limited by the availability of nitrogen or phosphorus; however, the response to nutrient limitations of microbial communities that control biogeochemical cycling critical to ecosystem function may be complex and may be associated with a range of processes, including methane production. We show that many, if not most, of the methanogens in the peatlands of the Florida Everglades possess thenifH gene and actively express it for N2 fixation coupled with methanogenesis. These findings indicate that archaeal N2 fixation would play crucial role in methane emissions and overall N cycle in subtropical wetlands suffering N limitation.

Methanogens Are Major Contributors to Nitrogen Fixation in Soils of the Florida Everglades